Combination of sPLA2 activity and OxPL/apoB cardiovascular risk factors for the diagnosis/prognosis of a cardiovascular disease/event

ABSTRACT

A method of identifying a subject having or at risk of having or developing a cardiovascular disease and/or a cardiovascular event, includes:
         measuring, in a sample obtained from the subject, at least two cardiovascular risk factors:
           a) sPLA2 activity and   b) oxidized phospholipids on apolipoprotein B-100 particles (OxPL/apoB),   
           combining the measurements, the combined value of sPLA2 activity and OxPL/apoB being indicative of having or a risk of having or developing a cardiovascular disease and/or cardiovascular event.

FIELD OF THE INVENTION

The invention relates to the use of a combination of sPLA2 activity andOxPL/apoB cardiovascular risk factors for the diagnosis/prognosis of acardiovascular disease/event or for the monitoring of a cardiovasculardisease.

BACKGROUND OF THE INVENTION

A key problem in treating vascular diseases is proper diagnosis. Oftenthe first sign of the disease is sudden death. For example,approximately half of all individuals who die of coronary artery diseasedie suddenly, Furthermore, for 40-60% of the patients who are eventuallydiagnosed as having coronary artery disease, myocardial infarction isthe first presentation of the disease. Unfortunately, approximately 40%of those initial events go unnoticed by the patient. Because of ourlimited ability to provide early and accurate diagnosis followed byaggressive treatment, cardiovascular diseases (CD) remain the primarycause of morbidity and mortality worldwide. Patients with CD represent aheterogeneous group of individuals, with a disease that progresses atdifferent rates and in distinctly different patterns. Despiteappropriate evidence-based treatments for patients with CD, recurrenceand mortality rates remain high. Also, the full benefits of primaryprevention are unrealized due to our inability to accurately identifythose patients who would benefit from aggressive risk reduction.

Whereas certain disease markers have been shown to predict outcome orresponse to therapy at a population level, they are not sufficientlysensitive or specific to provide adequate clinical utility in anindividual patient. As a result, the first clinical presentation formore than half of the patients with coronary artery disease is eithermyocardial infarction or death.

Physical examination and current diagnostic tools cannot accuratelydetermine an individual's risk for suffering a complication of CD. Knownrisk factors such as hypertension, hyperlipidemia, diabetes, familyhistory, and smoking do not establish the diagnosis of atherosclerosisdisease. Diagnostic modalities which rely on anatomical data (such ascoronary angiography, coronary calcium score, CT or MRI angiography)lack information on the biological activity of the disease process andcan be poor predictors of future cardiac events. Functional assessmentof endothelial function can be non-specific and unrelated to thepresence of atherosclerotic disease process, although some data hasdemonstrated the prognostic value of these measurements.

Individual biomarkers, such as the lipid and inflammatory markers, havebeen shown to predict outcome and response to therapy in patients withCD and some are utilized as important risk factors for developingatherosclerotic disease.

Nonetheless, up to this point, no single biomarker is sufficientlyspecific to provide adequate clinical utility for the diagnosis of CD inan individual patient. Therefore, there is a need for identifyingbiomarkers or cardiovascular risk factors or combination thereof thatprovides a more accurate diagnosis/prognosis of CD.

SUMMARY OF THE INVENTION

One object of the invention is a method of identifying a subject havingor at risk of having or developing a cardiovascular disease and/or acardiovascular event, comprising:

-   -   measuring, in a sample obtained from said subject, at least two        cardiovascular risk factors:        -   a) secretory phospholipases A2 (sPLA2) activity and        -   b) oxidized phospholipids on apolipoprotein B-100 particles            (OxPL/apoB),    -   combining said measurements, the combined value of sPLA2        activity and OxPL/apoB being indicative of having or a risk of        having or developing a cardiovascular disease and/or        cardiovascular event.

In one embodiment of the invention, said combined value of sPLA2activity and OxPL/apoB is compared to a reference value.

In another embodiment of the invention, said method further comprisesmeasuring at least one cardiovascular risk factor selected in the groupof Framingham Risk Score (FRS), C-reactive protein (CRP), IgM ICofapoB100 or IgM MDA-LDL, lipoprotein-associated phospholipaseA2(Lp-PLA2) and sPLA2 mass.

In one embodiment of the invention, said method is for identifying asubject having or at risk of having or developing a cardiovasculardisease and/or a cardiovascular event, said cardiovascular diseaseand/or cardiovascular event being Metabolic Syndrome, Syndrome X,atherosclerosis, atherothrombosis, coronary artery disease, stable andunstable angina pectoris, stroke, diseases of the aorta and its branches(such as aortic stenosis, thrombosis or aortic aneurysm), peripheralartery disease, peripheral vascular disease, cerebrovascular disease,and any acute ischemic cardiovascular event.

Another object of the invention is a method as described here above, formonitoring the efficacy of a treatment for a cardiovascular disease.

Another object of the invention is a kit for identifying whether asubject has or is at risk of having or developing a cardiovasculardisease and/or a cardiovascular event, comprising:

-   -   means for measuring sPLA2 activity and    -   means for measuring OxPL/apoB.

In one embodiment, said kit further comprises means for measuring atleast one cardiovascular risk factor selected in the group of FraminghamRisk Score (FRS), CRP, IgM IC of apoB100 or IgM MDA-LDL, Lp-PLA2 andsPLA2 mass.

DETAILED DESCRIPTION OF THE INVENTION

Definitions “Cardiovascular disease” or “arteriovascular disease” asdefined herein is a general term used to classify numerous conditionsaffecting the heart, heart valves, blood, and vasculature of the bodyand encompasses any disease affecting the heart or blood vessels,including, but not limited to, Metabolic Syndrome, Syndrome X,atherosclerosis, atherothrombosis, coronary artery disease, stable andunstable angina pectoris, stroke, diseases of the aorta and its branches(such as aortic stenosis, thrombosis or aortic aneurysm), peripheralartery disease, peripheral vascular disease, cerebrovascular disease,and including, without limitation, any acute ischemic cardiovascularevent. Arteriovascular disease as used herein is meant to most commonlyrefer to the ischemic or pro-ischemic disease, rather than generallyto-non-ischemic disease.

“Cardiovascular event” is used interchangeably herein with the term“cardiac event”, “acute arteriovascular event”, or “Arteriovascularevent” and refers to sudden cardiac death, acute coronary syndromes suchas, but not limited to, plaque rupture, myocardial infarction, unstableangina, as well as non-cardiac acute arteriovascular events such asblood clots of the leg, aneurysms, stroke and other arteriovascularischemic events where arteriovascular blood flow and oxygenation isinterrupted.

As used herein, “atherosclerosis” and “atherothrombosis” refer to acomplex arteriovascular inflammatory disease that develops in responseto multiple stimuli and cardiovascular risk factors, and is associatedwith systemic inflammation. Cells involved in the atheroscleroticprocess include vascular (endothelial and smooth muscle) cells,monocytes/macrophages, lymphocytes (T, B, NKT), dendritic cells, mastcells and platelets. They secrete or are stimulated by soluble factorsincluding peptides, glycoproteins, proteases and a set of cytokines Theyare involved in the perpetuation of the inflammatory response, theprogression and the destabilization of atherosclerosis, including plaqueerosion, rupture and thrombosis. Arteries harden and narrow due tobuildup of a material called “plaque” on their inner walls. As theplaque develops and increases in size, the insides of the arteries getnarrower (“stenosis”) and less blood can flow through them. Stenosis orplaque rupture may cause partial or complete occlusion of the affectedvasculature. Tissues supplied by the vasculature are thus deprived oftheir source of oxygenation (ischemia) and cell death(apoptosis/necrosis) can occur.

“CAD” or “coronary artery disease” is an arteriovascular disease whichoccurs when the arteries that supply blood to the heart muscle (thecoronary arteries) become atherosclerotic, calcified and/or narrowed.Eventually, blood flow to the heart muscle is reduced, and, becauseblood carries much-needed oxygen, the heart muscle is not able toreceive the amount of oxygen it needs, and often undergoes necrosis. CADis due to atherosclerosis and atherothrombosis of the blood vessels thatsupply the heart with oxygen-rich blood and leads to acute coronarysyndromes (ACS), myocardial infarction (heart attack), angina (stableand unstable). An estimated 13 million Americans are currently diagnosedwith CAD, with approximately 7 million being the survivors of past acuteevents. Over 1 million new acute CAD events occur each year, manyresulting in death. The lifetime risk of CAD after age 40 is 49 percentfor men and 32 percent for women. Subjects who are deemed clinically tobe at low risk or no risk for developing arteriovascular disease such asCAD often exhibit none or few of the traditional risk factors for thearteriovascular disease, but nevertheless may still be at risk for anacute arteriovascular event. Approximately 20% of all acute CAD eventsoccur in subjects with none of the traditional risk factors, and themajority of all acute CAD occur in subjects who have not been previouslydiagnosed with CAD. Often these subjects do not exhibit the symptoms ofan acute CAD event, i.e. shortness of breath and/or chest pain, untilthe actual occurrence of such an acute event. A substantial detectiongap remains for those who are at risk for an acute CAD event yet areasymptomatic, without traditional risk factors, or are currently deemedclinically to be at low risk and have not yet been diagnosed with CAD.“CVD” or “cerebrovascular disease” is an arteriovascular disease in theblood vessels that feed oxygen-rich blood to the face and brain, such asatherosclerosis and atherothrombosis. This term is often used todescribe “hardening” of the carotid arteries, which supply the brainwith blood. It is a common comorbid disease with CAD and/or PAD. It isalso referred to as an ischemic disease, or a disease that causes a lackof blood flow. CVD encompasses disease states such as “cerebrovascularischemia,” “acute cerebral infarction,” “stroke,” “ischemic stroke,”“hemorrhagic stroke,” “aneurysm,” “mild cognitive impairment (MCI)” and“transient ischemic attacks” (TIA). Ischemic CVD is believed to closelyrelate to CAD and PAD; non-ischemic CVD may have multiplepathophysiologies. An estimated 5 million Americans are the survivors ofpast diagnosed acute CVD events, with an estimated 700 thousand acuteCVD events occurring each year. As disclosed herein, subjects deemed tobe at low risk or no risk of CVD based on clinical assessments oftraditional arteriovascular disease risk factors, or without symptomssuch as TIAs, MCI or severe headache, may still be at risk for an acuteCVD event.

“PAD” or “peripheral artery disease” encompasses disease states such asatherosclerosis and atherothrombosis that occur outside the heart andbrain. It is a common comorbid disease with CAD. Subjects who are deemedto be at low risk or no risk of PAD based upon an assessment oftraditional risk factors of PAD (or arteriovascular disease), or who areasymptomatic for PAD or an arteriovascular disease may nevertheless beat risk for an arteriovascular event, even in the absence ofclaudication. Claudication can be defined as pain or discomfort in themuscles of the legs occurring due to a decreased amount of blood flowingto a muscle from narrowing of the peripheral arteries, producingischemia and often arterial occlusion, causing skeletal muscle and limbnecrosis. The pain or discomfort often occurs when walking anddissipates under resting conditions (intermittent claudication). Pain,tightness, cramping, tiredness or weakness is often experienced as aresult of claudication. PAD not only causes the hemodynamic alterationscommon in CAD, but also results in metabolic changes in skeletal muscle.When PAD has progressed to severe chronic and acute peripheral arterialocclusion, surgery and limb amputation often become the sole therapeuticoptions. PAD is widely considered to be an underdiagnosed disease, withthe majority of confirmed diagnoses occurring only after symptoms aremanifested, or only with other arteriovascular disease, and irreversiblearteriovascular damage due to such ischemic events has already occurred.

“Cardiovascular Risk Factor” encompasses one or more biomarker whoselevel is changed in subjects having a cardiovascular disease orpredisposed to developing a cardiovascular disease, or at risk of acardiovascular event.

“Risk” in the context of the present invention, relates to theprobability that an event will occur over a specific time period, as inthe conversion to arteriovascular events, and can mean a subject's“absolute” risk or “relative” risk. Absolute risk can be measured withreference to either actual observation post-measurement for the relevanttime cohort, or with reference to index values developed fromstatistically valid historical cohorts that have been followed for therelevant time period. Relative risk refers to the ratio of absoluterisks of a subject compared either to the absolute risks of low riskcohorts or an average population risk, which can vary by how clinicalrisk factors are assessed. Odds ratios, the proportion of positiveevents to negative events for a given test result, are also commonlyused (odds are according to the formula p/(1-p) where p is theprobability of event and (1-p) is the probability of no event) tono-conversion.

“Risk evaluation,” or “evaluation of risk” in the context of the presentinvention encompasses making a prediction of the probability, odds, orlikelihood that an event or disease state may occur, the rate ofoccurrence of the event or conversion from one disease state to another,i.e., from a normal condition to an arteriovascular condition or to oneat risk of developing an arteriovascular event, or from at risk of anarteriovascular event to a more stable arteriovascular condition. Riskevaluation can also comprise prediction of future clinical parameters,traditional laboratory risk factor values, or other indices ofarteriovascular disease, such as coronary calcium scores, other imagingor treadmill scores, passive or provocative tesing results,arteriovasculature percentage stenosis or occlusion and othermeasurements of plaque burden and activity, either in absolute orrelative terms in reference to a previously measured population. Themethods of the present invention may be used to make continuous orcategorical measurements of the risk of conversion to arteriovasculardisease and events, thus diagnosing and defining the risk spectrum of acategory of subjects defined as being at risk for an arteriovascularevent. In the categorical scenario, the invention can be used todiscriminate between normal and other subject cohorts at higher risk forarteriovascular events. In other embodiments, the present invention maybe used so as to discriminate those at risk for developing anarteriovascular event from those having arteriovascular disease, orthose having arteriovascular disease from normal.

A “sample” in the context of the present invention is a biologicalsample isolated from a subject and can include, by way of example andnot limitation, bodily fluids and/or tissue extracts such as homogenatesor solubilized tissue obtained from a subject. Tissue extracts areobtained routinely from tissue biopsy and autopsy material. Bodilyfluids useful in the present invention include blood, urine, saliva orany other bodily secretion or derivative thereof. As used herein “blood”includes whole blood, plasma, serum, circulating cells, constituents, orany derivative of blood.

“Clinical parameters or indicia” encompasses all non-sample ornon-analyte biomarkers of subject health status or othercharacteristics, such as, without limitation, age (Age), ethnicity(RACE), gender (Sex), diastolic blood pressure (DBP) and systolic bloodpressure (SBP), family history (FamHX), height (HT), weight (WT), waist(Waist) and hip (Hip) circumference, body-mass index (BMI), as well asothers such as Type I or Type II Diabetes Mellitus or GestationalDiabetes Mellitus (DM or GDM, collectively referred to here asDiabetes), and resting heart rate.

A “subject” in the context of the present invention is preferably amammal. The mammal can be a human, non-human primate, mouse, rat, dog,cat, horse, or cow, but are not limited to these examples. Mammals otherthan humans can be advantageously used as subjects that represent animalmodels of arteriovascular disease or arteriovascular events. A subjectcan be male or female. A subject can be one who has been previouslydiagnosed or identified as having arteriovascular disease or anarteriovascular event, and optionally has already undergone, or isundergoing, a therapeutic intervention for the arteriovascular diseaseor arteriovascular event. Alternatively, a subject can also be one whohas not been previously diagnosed as having arteriovascular disease. Forexample, a subject can be one who exhibits one or more risk factors forarteriovascular disease, or a subject who does not exhibitarteriovascular risk factors, or a subject who is asymptomatic forarteriovascular disease or arteriovascular events. A subject can also beone who is suffering from or at risk of developing arteriovasculardisease or an arteriovascular event.

A “formula,” “algorithm,” or “model” is any mathematical equation,algorithmic, analytical or programmed process, or statistical techniquethat takes one or more continuous or categorical inputs (herein called“parameters”) and calculates an output value, sometimes referred to asan “index” or “index value.” Non-limiting examples of “formulas” includesums, ratios, and regression operators, such as coefficients orexponents, biomarker value transformations and normalizations(including, without limitation, those normalization schemes based onclinical parameters, such as gender, age, or ethnicity), rules andguidelines, statistical classification models, and neural networkstrained on historical populations. Of particular use in combiningCardiovascular Risk Factor and other biomarkers are linear andnon-linear equations and statistical classification analyses todetermine the relationship between levels of Cardiovascular Risk Factordetected in a subject sample and the subject's risk of cardiovasculardisease. In panel and combination construction, of particular interestare structural and synactic statistical classification algorithms, andmethods of risk index construction, utilizing pattern recognitionfeatures, including established techniques such as cross-correlation,Principal Components Analysis (PCA), factor rotation, LogisticRegression (LogReg), Linear Discriminant Analysis (LDA), EigengeneLinear Discriminant Analysis (ELDA), Support Vector Machines (SVM),Random Forest (RF), Recursive Partitioning Tree (RPART), as well asother related decision tree classification techniques, ShrunkenCentroids (SC), StepAIC, Kth-Nearest Neighbor, Boosting, Decision Trees,Neural Networks, Bayesian Networks, Support Vector Machines, and HiddenMarkov Models, among others. Other techniques may be used in survivaland time to event hazard analysis, including Cox, Weibull, Kaplan-Meierand Greenwood models well known to those of skill in the art.

“Measuring” or “measurement,” or alternatively “detecting” or“detection,” means assessing the presence, absence, quantity or amount(which can be an effective amount) of either a given substance within aclinical or subject-derived sample, including the derivation ofqualitative or quantitative concentration levels of such substances, orotherwise evaluating the values or categorization of a subject'snon-analyte clinical parameters.

THE INVENTION

One object of the invention is a method of identifying a subject havingor at risk of having or developing a cardiovascular disease and/or acardiovascular event, comprising:

-   -   measuring, in a sample obtained from said subject, at least two        cardiovascular risk factors:        -   a) sPLA2 activity and        -   b) oxidized phospholipids on apolipoprotein B-100 particles            (OxPL/apoB),    -   combining said measurements, the combined value of sPLA2        activity and OxPL/apoB being indicative of having or a risk of        having or developing a cardiovascular disease and/or        cardiovascular event.

According to the invention, combining said measurements using astatistical analysis results in obtaining a combined value, which isindicative of having or a risk of having or developing a cardiovasculardisease and/or cardiovascular event.

In one embodiment of the invention, the subject may be a substantiallyhealthy subject, which means that the subject has not been previouslydiagnosed or identified as having or suffering from a cardiovasculardisease, or that has not developed a cardiovascular event. In anotherembodiment, the subject may also be one that is asymptomatic for thecardiovascular disease. As used herein, an “asymptomatic” subject refersto a subject that do not exhibit the traditional symptoms of acardiovascular disease or event, including, but not limited to, chestpain and shortness of breath for CAD, claudication for PAD, and TIAS,MCI and severe headache for CVD.

In another embodiment of the invention, the subject may be one that isat risk of having or developing a cardiovascular disease orcardiovascular event, as defined by clinical and biological indicia suchas for example: age, gender, LDL concentration, HDL concentration,triglyceride concentration, blood pressure, body mass index, CRPconcentration, coronary calcium score, waist circumference, tobaccosmoking status, previous history of cardiovascular disease, familyhistory of cardiovascular disease, heart rate, fasting insulinconcentration, fasting glucose concentration, diabetes status, and useof high blood pressure medication.

In another embodiment of the invention, the subject may be one that hasbeen previously diagnosed or identified for a cardiovascular disease orcardiovascular event, such as for example chronic ischemic disorderswithout myocardial necrosis (for example stable or effort anginapectoris), acute ischemic disorders without myocardial necrosis (forexample unstable angina pectoris), ischemic disorders with myocardialnecrosis (for example ST segment evaluation myocardial infarction ornon-ST segment elevation myocardial infarction).

Tissue ischemia is often defined in relative terms and occurs when theneeds in oxygen exceed the delivery of oxygen to tissues. There is animbalance between tissue (myocardial for example) oxygen demands andsupply. This condition of oxygen deprivation may be accompanied byinadequate removal of metabolites consequent to reduced perfusion.Myocardial ischemia can be diagnosed clinically (chest pain forexample), biologically (increase in myeloperoxidase activity forexample), metabolically, using scintigraphy, by analyzing regional wallmotion disorders or by use of an electrocardiogram (typicalmodifications of the ST segment, upper or lower ST segment deviation,typical changes in T wave such as T wave inversion or steep symmetric orhigh amplitude positive T waves). Silent ischemia is typically diagnosedusing scintigraphy or a 24h electrocardiogram recording.

Stable and effort angina is typically manifested by a chest pain duringexercise and slowly recovers at rest. It usually reflects tissueischemia during exercise.

Unstable angina is a recent increase in the frequency and/or severity ofstable angina, a first episode of angina, or an angina at rest.

Myocardial necrosis is typically diagnosed by an increase in myocardialenzymes (for example troponin I, troponin T, CPK) in the circulatingblood.

In another embodiment of the invention, the subject may be one that whoshows an improvement in cardiovascular risk factors as a result oftreatments and/or therapies for cardiovascular diseases. Suchimprovements include a reduction in body mass index, a reduction intotal cholesterol, a reduction in LDL levels, an increase in HDLClevels, a reduction in systolic and/or diastolic blood pressure, orother aforementioned risk factor or combinations thereof.

In one embodiment of the invention, no onset of ischemic symptom hasbeen diagnosed in the subject. Myocardial ischemia can be diagnosedclinically (chest pain for example), biologically (increase inmyeloperoxidase activity for example), metabolically using scintigraphy,by analysing regional wall motion disorders or by use of anelectrocardiogram (typical modifications of the ST segment, upper orlower ST segment deviation, typical changes in T wave such as T waveinsertion or steep symmetric or high amplitude positive T waves).

In another embodiment, an onset of ischemic symptoms has been diagnosedin the subject.

In one embodiment of the invention, the sample used to measure sPLA2activity and OxPL/apoB and optionally other cardiovascular risk factorsis a blood sample, whole blood, plasma, serum.

According to the invention, sPLA2 activity is measured in said sample.According to the invention, the measure of sPLA2 activity can beperformed by a fluorimetric assay according to Radvanyi et al. (1989Anal Biochem 177: 103-9) as modified by Pernas et al. (1991 BiochemBiophys Res Commun 178: 1298-1305), all incorporated by reference.

In particular, the following assay is used. The1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphomethanol sodiumsalt (Interchim, Montlucon, France) is used as a substrate for sPLA2.The hydrolysis of this substrate by sPLA2 yields 1-pyrenedecanoic acid,which emits fluorescence at 397 nm. A volume (E) of 0.03 ml of thealiquoted plasmas is mixed with 5 nmol of substrate in presence of a 10mM Tris-HCL pH 8.7, 0.1% albumin, 10 mM CaCl2 in a total volume of 2.5ml, and fluorescence (F) is measured at 397 nm after one minute. 100%hydrolysis of the substrate is obtained with 0.1 U of bee venom PLA2(Sigma Chemical Co., France) during one minute, the value of thefluorescence at the end of the one minute reaction (Fmax) thuscorresponds to an activity of 2 nmoles/min/ml (Vmax). The activity (A)of the sample (expressed in nmol/ml/min) is given by the followingformula: A=(Vmax*F)/(E*Fmax).

The samples are diluted when substrate hydrolysis is above 50%. Thehydrolysis of substrate in the absence of plasma is used as negativecontrol and deduced from PLA2 activity. All samples are tested induplicate. The minimum detectable activity and detection limit is 0.10nmole/min/ml and the intra and interassay coefficient of variation islower than 10%.

According to the invention, the measure of sPLA2 activity can beperformed by an improved fluorimetric assay, using an automatedfluorimetric measurement, with a small sample volume, a modifiedsubstrate/enzyme ratio (10 nmoles/U instead of 50 nmoles/U) and athermostat ruled at 30° C., providing a higher precision and sensitivity(2,7% <within batch coefficient of variation (CV)<3.2% and between batchCV=5.7%) than the previous method (within batch CV<10% and between batchCV<10%) and a substantially shorter time to complete the assay. Inparticular, the following assay is used for automated measurement. The 1-hexadecanoyl-2-(1 -pyrenedecanoyl)-sn-glycero-3-phosphomethanol sodiumsalt (Interchim, Montlucon, France) is used as a substrate for sPLA2.The hydrolysis of this substrate by sPLA2 yields 1-pyrenedecanoic acid,which emits fluorescence at 405 nm. Briefly, 1 nmol of fluorescentsubstrate in 0.2 ml of buffer substrate (10 mM Tris-HCL pH 8.7, 0.1%albumin, 10 mM CaCl2) was automatically distributed in Black Maxisorpmicrotitration plate (96 wells). Because the self-quenching propertiesof the substrate, a low fluorescence is firstly recorded (Fmin) in aFluostar Optima fluorimeter equipped with a stirring device andthermostat ruled at 30° C. The addition of 30 μl (100 U/mL) of bee venomPLA2 (Sigma Chemical Co., France) leads to a rapid hydrolysis of allsubstrate (100% of hydrolysis) and an increase in fluorescence to amaximal value (Fmax), corresponding to an activity of 5 nmol/ml. Todetermine the sPLA2 activity in unknown blood samples, 30 μl of sera (E)were automatically distributed and added to the substrate mixture andthe fluorescence was recorded at one minute (F). A two-point procedurewas used to measure the corrected fluorescence intensity of each sampleand to evaluate the enzymatic activity (expressed in nmol/min/ml). Allsamples were tested in duplicate. The activity (A) of the sample(expressed in nmol/ml/min) is given by the following formula:A=(Vmax*F)/(E*Fmax).

The hydrolysis of the substrate in the absence of serum is used asnegative control and deduced from PLA2 activity. All samples are testedin duplicate. Unless otherwise mentioned, all the numerical values givenherein for serum sPLA2 activity are measured according to the abovedefined assay for automated measurement.

The phospholipase that can be used to perform the assay is a secretoryphospholipase or a phospholipase with a known activity and preferably abee venom phospholipase.

In another embodiment, sPLA2 activity may be determined by a processbased on a fluorimetric assay comprising contacting a biological samplecontaining said sPLA2 and taken from said patient, with a substrate at aconcentration from 1 nM to 15 nM, the serum sample volume being from 5μl to 50 μl and the substrate volume being from 100 μlto 300 μl, at atemperature range from about 15° C. to about 40° C. and preferably 30°C. The phospholipase used could be a phospholipase from bee venom orsnake venom like Naja venom, preferably bee venom. It could be arecombinant phospholipase from any species. This assay is described inexample 2 of W02008/015546, which is incorporated by reference. Theadvantage of this method is the small sample volume of substrate usedand the thermostating, providing a higher precision and sensitivity.

Alternatively, a variant of the automated fluorimetric measurement asdefined above can be used, which enables to alleviate imprecision whichmight result from a non-specific increase in fluorescence intensity dueto other factors in the sample, thus interfering with the measure ofsPLA2 activity. This method only differs from the above-definedautomated fluorimetric measurement method in that the following formulais used for determining sPLA2 activity:A=F*s/[(Fmax−Fmin)*V]wherein:

-   -   A represents sPLA2 activity expressed in nmol/min/ml;    -   s represents the quantity of substrate expressed in nmol        (usually 1 nmol in a volume of 200 μl of working solution);    -   V represents the sample volume expressed in ml (usually from        0.30 to 0.50 ml);    -   (Fmax-Fmin) represents the difference between the maximal        fluorescence signal at the end of the reaction in the presence        of PLA2 from bee venom and the negative control;    -   F represents the initial slope, within linear range, of the        curve representing fluorescence emission as a function of time,        expressed in min⁻¹.

This variant of the automated fluorimetric measurement is described inexample 4 of WO2008/015546, which is incorporated by reference.

According to the invention, OxPL/apoB is measured in said sample.

Methods for measuring the ratio OxPL/apoB is described in US2006/177435,which is incorporated by reference. Said methods are based on thedetermination of OxPL level in the sample, the determination of apoBlevel in the sample, and then calculating the ratio OxPL/apoB.

In one embodiment, the level of OxPL and the level of apoB in the sampleobtained from the subject are measured with two or more differentbiomolecules. The first biomolecule specifically interacts with OxPL andthe second biomolecule specifically interacts with apoB. In someaspects, the biomolecules are antibodies, such as, for example,monoclonal antibodies. The antibody that interacts with OxPL may be, forexample, E06 or DLH3.

In other aspects, the biomolecules are antigens. In some embodiments,the biomolecules are immobilized to form an array comprising a first setof a plurality of the first biomolecule and a second set of a pluralityof the second biomolecule.

Exemplary oxidized phospholipid include oxidized forms of1-palmitoyl-2-arachidonoyl-sn-glycero-3-phos-phorylcholine (Ox-PAPC),1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphoryl-choline (POVPC),1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC),1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC),oxidized 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholin-e(Ox-SAPC), 1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine(SOVPC, 1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (SGPC),1-stearoyl-2-epoxyisopro stane-sn-glycero-3-phosphorylcholine (SEIPC),1-stearoyl-2-arachidonyl-sn-glycero-3-phosphorylethanolamine(Ox-SAPE),1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylethanolamine(SOVPE),1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylethanolamine(SGPE), and1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylethanolamine(SEIPE).

As used herein, the terms “biological molecules” and “biomolecules” maybe used interchangeably. These terms are meant to be interpretedbroadly, and generally encompass polypeptides, peptides,oligosaccharides, polysaccharides, oligopeptides, proteins,oligonucleotides, and polynucleotides. Oligonucleotides andpolynucleotides include, for example, DNA and RNA, e.g., in the form ofaptamers. Biomolecules also include organic compounds, organometalliccompounds, salts of organic and organometallic compounds, saccharides,amino acids, and nucleotides, lipids, carbohydrates, drugs, steroids,lectins, vitamins, minerals, metabolites, cofactors, and coenzymes.Biomolecules further include derivatives of the molecules described. Forexample, derivatives of biomolecules include lipid and glycosylationderivatives of oligopeptides, polypeptides, peptides, and proteins, suchas antibodies. Further examples of derivatives of biomolecules includelipid derivatives of oligosaccharides and polysaccharides, e.g.,lipopolysaccharides. An exemplary biochemical test for identifyingspecific proteins, such as OxPL and apoB, employs a standardized testformat, such as the Enzyme Linked Immunosorbent Assay or ELISA test,although the information provided herein may apply to the development ofother biochemical or diagnostic tests and is not limited to thedevelopment of an ELISA test (see, e.g., Molecular Immunology: ATextbook, edited by Atassi et al. Marcel Dekker Inc., New York and Basel1984, for a description of ELISA tests). It is understood thatcommercial assay enzyme-linked immunosorbant assay (ELISA) kits forvarious plasma constituents are available.

Improved methods for measuring the ratio OxPL/apoB is described inWO01/88547, which is incorporated by reference. Said improved methodsare intended to standardize the assay by using a phosphorylcholine (PC).According to said method, an immunoassay can be performed either byfirst capturing the LDL on a microtiter well by use of an antibody thatbinds both oxidized and nonoxidized LDL (e. g. anti-apoB), and thendetection of the OxLDL by a labelled E06 antibody. Alternatively, E06antibody can be bound to the bottom of the microtiter well and theamount of OxLDL bound determined by the use of labeled anti-LDLantibody. OxLDL could also be used to coat the microtiter wells andvarious concentrations of patient sera, putatively containing OxLDL,could be mixed with a constant, limiting amount of labeled (e. g.biotinylated) E06 or T15 to compete for binding to the OxLDL on theplate. For each assay, under standard conditions, a standard curve couldbe developed using PC as a competing agent. Alternatively, a parallelset of reactions can be run using PC as a source of competing agentrather than patient sera. The PC can be used alone, or linked to acarrier protein, such as bovine serum albumin (BSA) or keyhole limpethemocyanin (KLH).

According to the invention, the measurements obtained for sPLA2 activityand OxPL/apoB and optionally other cardiovascular risk factors arecombined in statistical analysis, wherein the combined value of sPLA2activity and OxPL/apoB and optionally other cardiovascular risk factorsis indicative of having or a risk of having or developing acardiovascular disease and/or cardiovascular event.

As the skilled artisan will appreciate, there are many ways to use themeasurements of two or more risk factors in order to improve thediagnostic/prognostic question under investigation.

In a quite simple, but nonetheless often effective approach, a positiveresult is assumed if a sample is positive for at least one of themarkers investigated. This may be for example the case when diagnosingan infectious disease, like AIDS. Frequently, however, a combination ofrisk factors is evaluated. Preferably the measurements obtained forsPLA2 activity and OxPL/apoB and optionally other cardiovascular riskfactors are mathematically combined and the combined value is correlatedto the underlying diagnostic/prognostic question. Risk factormeasurements may be combined by any appropriate state of the artmathematical method. Well-known mathematical methods for correlating amarker combination to a disease employ methods like, Discriminantanalysis (DA) (i.e. linear-, quadratic-, regularized-DA), Kernel Methods(i.e. SVM), Nonparametric Methods (i.e. k-Nearest-Neighbor Classifiers),PLS (Partial Least Squares), Tree-Based Methods (i.e. Logic Regression,CART, Random Forest Methods, Boosting/Bagging Methods), GeneralizedLinear Models (i.e. Logistic Regression), Principal Components basedMethods (i.e. SIMCA), Generalized Additive Models, Fuzzy Logic basedMethods, Neural Networks and Genetic Algorithms based Methods. Theskilled artisan will have no problem in selecting an appropriate methodto evaluate a marker combination of the present invention.

Preferably the method used in correlating the risk factors combinationof the invention to the risk of having or being at risk of having acardiovascular disease or cardiovascular event is selected from DA (i.e.Linear-, Quadratic-, Regularized Discriminant Analysis), Kernel Methods(i.e. SVM), Nonparametric Methods (i.e. k-Nearest-Neighbor Classifiers),PLS (Partial Least Squares), Tree-Based Methods (i.e. Logic Regression,CART, Random Forest Methods, Boosting Methods), or Generalized LinearModels (i.e. Logistic Regression). Details relating to these statisticalmethods are found in the following references: Ruczinski, I., KooperbergC., LeBlanc, M., Logic regression, J. of Computational and GraphicalStatistics, 12 (2003) 475-511; Friedman, J. H. , RegularizedDiscriminant Analysis, J. of the American Statistical Association, 84(1989) 165-175; Trevor Hastie, Robert Tibshirani and Jerome Friedmann,The Elements of Statistical Learning, Springer Verlag, 2001; Breiman,L., Friedman, J. H., Olshen, R. A., Stone, C. J. (1984) Classificationand regression trees, California: Wadsworth; Breiman, L., RandomForests, Machine Learning, 45 (2001) 5-32; Pepe, M. S., The StatisticalEvaluation of Medical Tests for Classification and Prediction, OxfordStatistical Science Series, 28 (2003); and Duda, R. O., Hart, P. E.,Stork, D. G., Pattern Classification, Wiley Interscience, 2nd Edition(2001).

In one embodiment of the invention, an optimized multivariate cut-offfor the underlying combination of risk factors is used to discriminatestate A from state B, e.g. diseased from substantially healthy. In thistype of analysis, the risk factors are no longer independent but form arisk factor panel. Combining the measurements of sPLA2 activity andOxPL/apoB does significantly improve the diagnostic/prognostic accuracyfor cardiovascular disease and/or cardiovascular event as compared tosubstantially healthy subjects or as compared to subjects which havebeen diagnosed for a cardiovascular disease or event.

In one embodiment of the invention, the statistical analysis of themeasurements of sPLA2 activity and OxPL/apoB and optionally othercardiovascular risk factors is based on the determination of odds ratios(OR) using standard procedures. An odds ratio is calculated by dividingthe odds in the test group by the odds in the control group. The odds ofan event are calculated as the number of events divided by the number ofnon-events. If the odds of an event are greater than one the event ismore likely to happen than not (the odds of an event that is certain tohappen are infinite); if the odds are less than one the chances are thatthe event won't happen (the odds of an impossible event are zero). Ingeneral, the strength of association is reported as odds ratios (OR)(with 95% lower (LCL) and upper (UCL) confidence limit), indicating thefactor by which the risk of having a disease or being at risk of havingor developing a disease is increased (OR>1). The 95% confidence interval(95% Cl) is the range of numerical values in which we can be confident(to a computed probability, here 95%) that the population value beingestimated will be found. Confidence intervals indicate the strength ofevidence; where confidence intervals are wide, they indicate lessprecise estimates of effect. The larger the trial's sample size, thelarger the number of outcome events and the greater becomes theconfidence that the true relative risk reduction is close to the valuestated. Thus the confidence intervals get narrower and “precision” isincreased. To confidently accept a calculated OR as reliable, importantor clinically significant, the lower boundary of the confidenceinterval, or lower confidence limit, should be >1 if the OR>1, or theupper boundary of the confidence interval should be <1 if the OR<1.

In another embodiment of the invention, accuracy of adiagnostic/prognostic method is best described by its receiver-operatingcharacteristics (ROC) (see especially Zweig, M. H., and Campbell, G.,Clin. Chem. 39 (1993) 561-577). The ROC graph is a plot of all of thesensitivity/specificity pairs resulting from continuously varying thedecision thresh-hold over the entire range of data observed.

The clinical performance of a laboratory test depends on its diagnosticaccuracy, or the ability to correctly classify subjects into clinicallyrelevant subgroups. Diagnostic accuracy measures the test's ability tocorrectly distinguish two different conditions of the subjectsinvestigated. Such conditions are for example health and disease.

In each case, the ROC plot depicts the overlap between the twodistributions by plotting the sensitivity versus 1—specificity for thecomplete range of decision thresholds. On the y-axis is sensitivity, orthe true-positive fraction [defined as (number of true-positive testresults)/(number of true-positive + number of false-negative testresults)]. This has also been referred to as positivity in the presenceof a disease or condition. It is calculated solely from the affectedsubgroup. On the x-axis is the false-positive fraction, or 1—specificity[defined as (number of false-positive results)/(number oftrue-negative + number of false-positive results)]. It is an index ofspecificity and is calculated entirely from the unaffected subgroup.Because the true- and false-positive fractions are calculated entirelyseparately, by using the test results from two different subgroups, theROC plot is independent of the prevalence of disease in the sample. Eachpoint on the ROC plot represents a sensitivity/1-specificity paircorresponding to a particular decision threshold. A test with perfectdiscrimination (no overlap in the two distributions of results) has anROC plot that passes through the upper left corner, where thetrue-positive fraction is 1.0, or 100% (perfect sensitivity), and thefalse-positive fraction is 0 (perfect specificity). The theoretical plotfor a test with no discrimination (identical distributions of resultsfor the two groups) is a 45° diagonal line from the lower left corner tothe upper right corner. Most plots fall in between these two extremes.(If the ROC plot falls completely below the 45° diagonal, this is easilyremedied by reversing the criterion for “positivity” from “greater than”to “less than” or vice versa). Qualitatively, the closer the plot is tothe upper left corner, the higher the overall accuracy of the test.

One convenient goal to quantify the diagnostic accuracy of a laboratorytest is to express its performance by a single number. The most commonglobal measure is the area under the ROC plot. By convention, this areais always 0.5 (if it is not, one can reverse the decision rule to makeit so). Values range between 1.0 (perfect separation of the test valuesof the two groups) and 0.5 (no apparent distributional differencebetween the two groups of test values). The area does not depend only ona particular portion of the plot such as the point closest to thediagonal or the sensitivity at 90% specificity, but on the entire plot.This is a quantitative, descriptive expression of how close the ROC plotis to the perfect one (area=1.0).

In one embodiment of the invention, the combined value of sPLA2 activityand OxPL/apoB and optionally other risk factors is compared to areference value.

In one embodiment, the reference value may be an index value or may bederived from one ore more risk prediction algorithms or computed indicesfor the cardiovascular disease and/or cardiovascular event. A referencevalue can be relative to a number or value derived from populationstudies, including without limitation, such subjects having similar bodymass index, total cholesterol levels, LDL/HDL levels, systolic ordiastolic blood pressure, subjects of the same or similar age range,subjects in the same or similar ethnic group, subjects having familyhistories of atherosclerosis, atherothrombosis, or CAD, PAD, or CVD, orrelative to the starting sample of a subject undergoing treatment for anarteriovascular disease, such as atherosclerosis, atherothrombosis, CAD,PAD, or CVD.

Such reference values can be derived from statistical analyses and/orrisk prediction data of populations obtained from mathematicalalgorithms and computed indices of arteriovascular disease, such as butnot limited to, algorithms reported in the Framingham Study, NCEP/ATPIII, among others. Cardiovascular Risk Factor reference value can alsobe constructed and used using algorithms and other methods ofstatistical and structural classification.

In one embodiment of the present invention, the reference value isderived from the combination of OxPL/apoB and sPLA2 activity andoptionally others cardiovascular risk factors in a control samplederived from one or more subjects who are substantially healthy asdefined here above. Such subjects who are substantially healthy lacktraditional risk factors for a cardiovascular disease: for example,those subjects have a serum cholesterol level less than 200 mg/dl,systolic blood pressure less than or equal to 120 mm Hg, diastolic bloodpressure less than or equal to 80 mm Hg, non-current smoker, no historyof diagnosed diabetes, no previously diagnosed acute coronary syndromeor hypertension, among other aforementioned other risk factors, or canbe verified by another invasive or non-invasive diagnostic test ofcardiovascular disease known in the art, such as but not limited to,electrocardiogram (ECG), carotid B-mode ultrasound (for intima-medialthickness measurement), electron beam computed tomography (EBCT),coronary calcium scoring, multi-slice high resolution computedtomography, nuclear magnetic resonance, stress exercise testing,angiography, intra- vascular ultrasound (IVUS), other contrast and/orradioisotopic imaging techniques, or other provocative testingtechniques.

In another embodiment, such subjects are monitored and/or periodicallyretested for a diagnostically relevant period of time (“longitudinalstudies”) following such test to verify continued absence fromcardiovascular disease or acute cardiovascular events (disease or eventfree survival). Such period of time may be one year, two years, two tofive years, five years, five to ten years, ten years, or ten or moreyears from the initial testing date for determination of the referencevalue. Furthermore, retrospective measurement of OxPL/apoB and sPLA2activity levels in properly banked historical subject samples may beused in establishing these reference values, thus shortening the studytime required, presuming the subjects have been appropriately followedduring the intervening period through the intended horizon of theproduct claim.

In another embodiment, a reference value can also be derived from thecombination of OxPL/apoB and sPLA2 activity and optionally othercardiovascular risk factors in a sample derived from one or more subjectwho (1) has been previously diagnosed or identified for a cardiovasculardisease or cardiovascular event by one of the above invasive ornon-invasive techniques, or who has suffered from an cardiovascularevent or plaque rupture, and (2) has not experienced a recurrentcardiovascular event.

In another embodiment, a reference value can also be derived from thecombination of OxPL/apoB and sPLA2 activity and optionally othercardiovascular risk factors in a sample derived from one or more subjectwho is at high risk for developing a cardiovascular event, or who is athigh risk for developing an atherosclerotic or atherothrombotic plaquerupture.

In another embodiment of the invention, a reference value can also bederived from the combination of OxPL/apoB and sPLA2 activity andoptionally other cardiovascular risk factors in a sample derived fromone or more subject who shows an improvement in cardiovascular riskfactors as a result of treatments and/or therapies for cardiovasculardiseases. Such improvements include a reduction in body mass index, areduction in total cholesterol, a reduction in LDL levels, an increasein HDLC levels, a reduction in systolic and/or diastolic blood pressure,or other aforementioned risk factor or combinations thereof.

In one embodiment of the invention, the reference value is an indexvalue or a baseline value. An index value or baseline value is derivedfrom one or more subjects who do not have a cardiovascular disease, suchas atherosclerosis, atherothrombosis, CAD, PAD, or CVD, or subjects whoare asymptomatic for a cardiovascular disease. A baseline value can alsobe derived from a subject who has shown an improvement in cardiovascularrisk factors (as a result of cardiovascular treatments or therapies.Such improvements include, without limitation, a reduction in body massindex, a reduction in total cholesterol, a reduction in LDL levels, anincrease in HDLC levels, a reduction in systolic and/or diastolic bloodpressure, or combinations thereof

In one embodiment of the invention, the method of the inventioncomprises combining sPLA2 activity and OxPL/apoB with clinical andbiological indicia such as age, a history of hypertension, diabetes,myocardial infarction, heart failure, coronary angiography orangioplasty, Killip class, ST-segment deviation, coronaryrevascularization (angioplasty or coronary artery bypass surgery), andcreatinine

In another embodiment of the invention, the method of the inventioncomprises:

-   -   measuring, in a sample obtained from said subject, at least two        cardiovascular risk factors:        -   a) sPLA2 activity and        -   b) oxidized phospholipids on apolipoprotein B-100 particles            (OxPL/apoB), and at least one cardiovascular risk factor            selected in the group of Framingham Risk Score (FRS), CRP,            IgM IC (IgM Immune Complexes) of apoB100 or IgM MDA-LDL (IgM            Malondialdehyde LDL), Lp-PLA2 activity and sPLA2 mass,    -   combining said measurements, the combined value being indicative        of having or a risk of having or developing a cardiovascular        disease and/or cardiovascular event.

According to said embodiment, FRS is calculated using a previouslyreported algorithm, which takes into account age, sex, totalcholesterol, HDL-C, systolic and diastolic blood pressure, smoking andthe presence of diabetes (Wilson P. W. et al., Circulation.1998;97:1837-1847, which is incorporated by reference).

According to said embodiment, CRP can be measured by methods known inthe art, such as a the method described in Arima et al (ArteriosclerThromb Vasc Biol. 2008 Jul; 28(7):1385-91) and Ridker et al. (NewEngland Journal of Medecine, 2000, 342:836-843), which are incorporatedby reference.

According to said embodiment, IgM IC of apoB100 and IgM MDA-LDL can bemeasured by methods known in the art such as according to Tsimikas et al(2004, Circulation, 110:1406-1412 and 2003 J. Am. Coll. Cardiol.41:360-370), which are incorporated by reference.

According to said embodiment, Lp-PLA-2 activity can be measured bymethods known in the art such as according to Kiechl et al. (2007,Atherioscler. Thromb. Vasc. Biol. 27:1788-1795), which is incorporatedby reference. For example, Lp-PLA2 activity may be measured using acommercially available kit (Azwell Inc) based on the method of Kosaka etal (2000 Clin. Chim. Acta 296:151-161, which is incorporated byreference). LpPLA2 activity may also be measured by the method describedin WO2005074604, which is incorporated by reference.

According to said embodiment, sPLA2 mass can be measured using animmunometric assay with a monoclonal antibody specific for sPLA2-IIA(Cayman Chemical Company).

In one embodiment, the method of the invention comprises the combinationof sPLA2 activity, OxPL/apoB and FRS measurements.

In another embodiment, the method of the invention comprises sPLA2activity, OxPL/apoB, FRS and CRP measurements.

In another embodiment, the method of the invention comprises sPLA2activity, OxPL/apoB, FRS and sPLA2 mass measurements.

According to the invention, the method as described here above is foridentifying whether a subject has or is at risk of having or developinga cardiovascular disease and/or a cardiovascular event.

In one embodiment of the invention, said cardiovascular disease and/orcardiovascular event is Metabolic Syndrome, Syndrome X, atherosclerosis,atherothrombosis, coronary artery disease, stable and unstable anginapectoris, stroke, diseases of the aorta and its branches (such as aorticthrombosis or aortic aneurysm), peripheral artery disease, peripheralvascular disease, cerebrovascular disease, and any acute ischemiccardiovascular event.

Optionally, subjects identified as having, or being at increased risk ofdeveloping a cardiovascular disease or cardiovascular event are chosento receive a therapeutic regimen to slow the progression of acardiovascular disease, or decrease or prevent the risk of developing acardiovascular disease or a cardiovascular event.

According to the invention, the method as described here above is formonitoring a cardiovascular disease or event in a subject in needthereof, said cardiovascular disease or event being Metabolic Syndrome,Syndrome X, atherosclerosis, atherothrombosis, coronary artery disease,stable and unstable angina pectoris, stroke, diseases of the aorta andits branches (such as aortic thrombosis or aortic aneurysm), peripheralartery disease, peripheral vascular disease, cerebrovascular disease,and any acute ischemic cardiovascular event.

In one embodiment of the invention, the method as described here aboveis for assessing the progression of a cardiovascular disease in asubject in need thereof.

In another embodiment of the invention, the method as described hereabove is for monitoring the effectiveness of a treatment for acardiovascular disease. The efficacy of the treatment will be reflectedby changes in the measurements of the cardiovascular risk factors. If atreatment has a desired effect, the measurements and thus the combinedvalue of the cardiovascular risk factors will be lower compared to themeasurements and combined value obtained before the treatment.

In another embodiment of the invention, the method as described hereabove is for selecting a treatment regimen for a subject diagnosed withor at risk for a cardiovascular disease.

Another object of the invention is a kit for identifying whether asubject has or is at risk of having or developing a cardiovasculardisease and/or a cardiovascular event, comprising:

-   -   means for measuring sPLA2 activity and    -   means for measuring OxPL/apoB.

In one embodiment, said kit further comprises means for combining themeasurements in order to obtain a combined value.

Said means for combining the measurements of the cardiovascular riskfactors are algorithms allowing statistical analysis such as DA (i.e.Linear-, Quadratic-, Regularized Discriminant Analysis), Kernel Methods(i.e. SVM), Nonparametric Methods (i.e. k-Nearest-Neighbor Classifiers),PLS (Partial Least Squares), Tree-Based Methods (i.e. Logic Regression,CART, Random Forest Methods, Boosting Methods), or Generalized LinearModels (i.e. Logistic Regression).

In another embodiment, said means for measuring sPLA2 activity are

-   -   a sPLA2 buffer,    -   a compound liable to be hydrolyzed by sPLA2, the hydrolytic        products of which can be directly or indirectly quantified,    -   a control sPLA2 activity sample.

According to said embodiment, the compound liable to be hydrolyzed bysPLA2 is a natural or non natural substrate of the enzyme. In case thehydrolysis products are not quantifiable by themselves, compounds whichcan react with these products and which yield quantifiable compounds canbe used, such a method is an indirect quantification. In general, thecompound liable to be hydrolysed by sPLA2 is a phospholipid or aphospholipid analogue comprising a fluorogenic or a chromogenic moiety.For example, said phospholipid is a glycerophospholipid which issubstituted in position 2 by a fluorescent acyl; such as1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphomethanol or thefluorescent acyl 1-pyerenedecanoyl, the substrate for horseradishperoxidase being for example 3,3′,5,5′ tetramethyl-benzidine (TMB).

Fluorescent acyls liable to be used according to the invention are forexample acyls substituted by fluorescent groups well known in the art,such as pyrene or fluoresceine. Alternatively, radioactiveglycerophospholipids can be used, such as glycerophospholipidssubstituted in position 2 by radioactive acyls or radioactivephosphatidyl ethanolamine. The control sPLA2 activity sample comprisesfor example bee venom sPLA2.

In another embodiment, said means for measuring OxPL/apoB are

-   -   an antibody that interacts specifically with OxPL such as for        example E06, T15 or DLH3 and    -   an antibody that interacts specifically with apoB such as for        example MB47,    -   optionally a control OxPL sample.

According to this embodiment, said control OxPL sample may be a samplecontaining a phosphorylcholine (PC). The PC can be used alone, or linkedto a carrier protein, such as bovine serum albumin (BSA) or keyholelimpet hemocyanin (KLH).

In another embodiment of the invention, said kit may further comprisemeans for measuring at least one cardiovascular risk factor selected inthe group of Framingham Risk Score (FRS), CRP, IgM IC of apoB100 or IgMMDA-LDL and LpPLA-2.

According to said embodiment, the Framingham Risk Score (FRS) isdetermined by method known in the art, such as the method described inWilson PW et al. (Circulation. 1998 May 12;97(18):1837-47) andD'Agostino et al. (JAMA: The Journal of the American MedicalAssociation. 2001;286:180-187), which are incorporated by reference.

According to said embodiment, said means for measuring CRP are forexample:

-   -   a CRP buffer    -   a monoclonal antibody that interacts specifically with CRP,    -   an enzyme-conjugated antibody specific for CRP,    -   a control CRP level.

According to said embodiment, said means for measuring IgM IC of apoB100o are for example:

-   -   an IgM of apoB100 buffer,    -   a chemiluminescent reagent    -   a monoclonal antibody specific for human apoB100,    -   a alkaline phosphatase-labelled anti human IgM,    -   a control IgM IC of apoB100 level sample.

According to said embodiment, said means for measuring IgM MDA-LDL arefor example:

-   -   an MDA-LDL buffer,    -   a chemiluminescent reagent    -   a alkaline phosphatase-labelled anti human IgM,    -   a control IgM MDA-LDL of apoB100 level sample.

According to said embodiment, said means for measuring Lp-PLA-2 are forexample:

-   -   a compound which reduces active thiol(s) in a sample, and    -   a substrate converted to a free thiol product in the presence of        enzymatically active Lp-PLA2.

Optionally, said means may further comprises an antibody that interactsspecifically with Lp-PLA2 such as for example the monoclonal antibody2C10, 4B4, B200, B501, 90D1E, 90E3A, 90E6C, 90G11D, or 90F2D.

For example, said compound which reduces active thiol(s) and saidsubstrate converted to a free thiol product in the presence ofenzymatically active Lp-PLA2 are described in WO2005074604, which isincorporated by reference.

According to said embodiment, said means for measuring sPLA2 mass arefor example:

-   -   a sPLA2 mass buffer    -   a monoclonal antibody that interacts specifically with        sPLA2-IIA,    -   an enzyme-conjugated antibody specific for sPLA2 IIA,    -   a control sPLA2 mass level.

DESCRIPTION OF THE FIGURES

FIG. 1: Baseline characteristics of study participants.

FIG. 2: Unadjusted odds ratios of incident coronary artery diseaseduring follow up according to combined tertiles of sPLA2 activity orLp-PLA2 activity and OxPL/apoB.

FIG. 3: Adjusted odds ratios of incident coronary artery disease duringfollow up according to combined tertiles of sPLA2 activity or Lp-PLA2activity and OxPL/apoB.

FIG. 4: Area under ROC for FRS and combination with at least one ofOxPL/apoB, Lp-PLA2 activity, sPLA2 activity and CRP.

FIG. 5: Odds ratios for CAD based on tertiles of OxPL/apoB and sPLA2activity, sPLA2 mass and Lp-PLA2.

The tertile cutoffs for OxPL/apoB are <1150 RLU, 1151-2249 RLU and >2249RLU, for sPLA2 activity levels <4.05 nmol/min per ml, 4.05-4.83 nmol/minper ml and >4.83 nmol/min per ml, for sPLA2 mass levels <6.80 mg/dl,6.80-11.29 mg/dl and >11.19 mg/dl, and for Lp-PLA2 activity <44.05nmol/min per ml, 44.05-56.23 nmol/min per ml and >56.23 nmol/min per ml.

FIG. 6: Area under ROC for FRS and combination with at least one ofOxPL/apoB, Lp-PLA2 activity, sPLA2 activity and CRP.

FIG. 7: Relationship between tertile groups of OxPL/apoB (<1150,1151-2249 and >2249 RLU, (A)), sPLA2 activity (<4.05, 4.05-4.83and >4.83 nmol/min per ml; (B)), sPLA2 mass (<6.80, 6.80-11.29and >11.19 mg/dl; (C)), Lp-PLA2 activity (<44.05, 44.05-56.23 and >56.23nmol/min per ml; (D)), and future CAD risk within each Framingham RiskScore Group. Framingham Risk Score was calculated as low risk (<10% riskof events over 10 years), moderate risk (10% to 20%), and high risk(>20%). The p-values in the figure represent comparison of each tertileof the respective biomarkers with the lowest tertile in the low FRScategory of each biomarker.

EXAMPLES

Methods

Detailed description of the European Prospective Investigation IntoCancer and Nutrition (EPIC-Norfolk) follow-up study have been publishedpreviously (Day N. et al., Br. J. Cancer, 1999; 80 Suppl. I: 95-103).Briefly, this prospective population study of 25,663 men and womenrecruited from age-sex registers of general practices in Norfolk, agedbetween 45 and 79 years, was designed to investigate dietary and otherdeterminants of cancer. The participants completed a baselinequestionnaire survey between 1993 and 1997, attended a clinic visit andwere followed up to November 2003, an average of about 6 years. Allindividuals have been flagged for death certification at the UK Officeof National Statistics, with vital status ascertained for the entirecohort. In addition, participants admitted to hospital were identifiedusing their unique National Health Service number by data linkage withthe East Norfolk Health Authority database, which identifies allhospital contacts throughout England and Wales for Norfolk residents.The study was approved by the Norwich Health Authority Ethics Committee,and all participants provided written informed consent.

Study Population (FIG. 1)

A nested-case control study was performed among participants in theEpic-Norfolk study. Case ascertainment has been described in detailelsewhere (Boekholdt, S. M., Circulation, 2004; 110:1418-1423. Briefly,25,663 healthy men and women, aged between 45 and 79 years, wererecruited from age—sex registers of general practices in Norfolk. Theparticipants completed a baseline questionnaire survey between 1993 and1997, attended a clinic visit and were followed for an average of 6years. Individuals who reported a history of heart attack or stroke atthe baseline visit were excluded. Case ascertainment has been describedpreviously. All individuals have been flagged for death certification atthe UK Office of National Statistics, with vital status ascertained forthe entire cohort. In addition, participants admitted to hospitals wereidentified using their National Health Service number by data linkagewith the East Norfolk Health Authority database, which identifies allhospital contacts throughout England and Wales for Norfolk residents.Participants were identified as having CAD during follow-up if they hada hospital admission and/or died with CAD as the underlying cause. CADwas defined as codes 410 to 414 according to the InternationalClassification of Diseases 9th revision. These codes encompass theclinical spectrum of CAD such as unstable angina, stable angina, andmyocardial infarction. Controls were study participants who remainedfree of any cardiovascular disease during follow-up. Were excluded allindividuals who reported a history of heart attack or stroke at thebaseline clinic visit. Two controls to each case by sex, age (within 5years) and time of enrolment (within 3 months) were matched. Data onnon-cardiac events has not been collected. The study was approved by theNorwich Health Authority Ethics Committee, and all participants providedwritten informed consent.

Study Measurements

Blood samples were stored at −80° C. at the Department of ClinicalBiochemistry, University of Cambridge. Serum levels of totalcholesterol, HDL-cholesterol (HDL-C) and triglycerides were measured onfresh samples with the RA 1000 (Bayer Diagnostics, Basingstoke, UK), andLDL-cholesterol (LDL-C) levels were calculated with the Friedewaldformula (Friedewald WT. et al., CHn Chem. 1972;18:499-502. CRP levelswere measured with a sandwich-type ELISA as previously described (BruinsP. et a \. Circulation. 1997;96:3542-3548).

Serum sPLA2 activity was measured by a selective fluorimetric assay ofRadvanyi et al. (Anal Biochem. 1989;177:103-109), as modified by Pernaset al. (Biophys Res Commun. 1991;178:1298-1305). The sPLA2 activity wasmeasured using fluorescent substrate1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3 phosphomethanol, sodiumsalt (Interchim, Montlucon, France) as previously described (Mallat Z.et al. J Am Coll Cardiol. 2005;46: 1249-1257). One hundred percenthydrolysis of the fluorescent substrate was measured using 0.1 unit PLA2from Bee venom (Sigma Chemical Co., France). The hydrolysis of substratein the absence of plasma was used as negative control and deduced fromPLA2 activity. All the samples were tested in duplicate and plasmaactivity was expressed as nmole/min/ml. The minimum detectable activitywas 0.10 nmole/min/ml. The imprecision of sPLA2 activity fluorimetricassay was determined by measurement of samples with low (1.25nmol/min/ml) and high (9.5 nmol/min/ml) sPLA2 activity. Within-batch CVsranged from 2.7% (low-activity sample) to 3.2% (high-activity sample)and the between-batch CV was 5.7%.

OxPL/apoB levels were measured with antibody E06 as in Tsimikas et al(Current Opinion in Lipidology, 2008, 19:369-377, which is incorporatedby reference).

All the samples were analyzed in random order to avoid systemic bias.Researchers and laboratory personnel were blinded to identifiableinformation, and could identify samples by number only.

Statistical Analysis

Baseline characteristics were compared between cases and matchedcontrols taking into account the matching between them. A mixed effectmodel was used for continuous variables and conditional logisticregression was used for categorical variables. Because triglycerides,CRP, OxPL/apoB, sPLA2 antigen levels and sPLA2 activity had a skeweddistribution, values were log-transformed before being used ascontinuous variables in statistical analyses; however, in the tables,untransformed medians and corresponding intertertile ranges are shown.

Tertiles were based on the distribution in the controls. Forsex-specific analyses, sex-specific tertiles were used, and for pooledanalyses, quartiles based on the sexes combined were used.

In addition, Pearson correlation coefficients were calculated to assessthe relationship between sPLA2 activity as a continuous variable andother continuous biomarkers of risk. Odds ratios and corresponding 95%confidence intervals (95% CIs) as an estimate of the relative risk ofincident CAD were calculated using conditional logistic regressionanalysis.

The lowest sPLA2 activity or the lowest Lp-PLA2 activity and the lowestOxPL/apoB tertiles were used as reference category.

Odds Ratios were adjusted for the following cardiovascular risk factors:body mass index, diabetes, systolic blood pressure, LDL-C, HDL-C, andsmoking (never, previous, current).

Odds Ratios were also adjusted for the FRS score. The Framingham RiskScore was calculated using a previously reported algorithm, which takesinto account age, sex, total cholesterol, HDL-C, systolic and diastolicblood pressure, smoking and the presence of diabetes (Wilson P. W. etal., Circulation. 1998;97:1837-1847). Statistical analyses wereperformed using SPSS software (version 12.0.1; Chicago, III).A P-value<0.05 was considered to indicate statistical significance.

Results

Combined Measurements of sPLA2 Activity and OxPL/apoB to Assess the Riskof Incident CAD

We selected cases that developed CHD during follow-up and selectedcontrols that remained free of cardiovascular disease, and were matchedto cases by sex, age and enrolment time. The risk of CHD events wassignificantly potentiated by elevated activity of sPLA2 and Lp-PLA2 andby elevated OxPL/apoB. People in the highest tertiles of both OxPL/apoBand LpPLA2 activity had an odds ratio of 2.22 (1.51-3.27) and patientsin the highest tertiles of both OxPL/apoB and sPLA2 activity had an oddsratio of 4.34 (2.84-6.64) (p<0.0001 for both compared to those in lowesttertiles for both) (FIG. 2 and FIG. 3 for OR's adjusted for FRS score).

Results shown in FIG. 5 are based on 763 cases and 1397 controls whereinall subject characteristics listed in table 1 were available.

FIG. 5 shows that subjects in the highest tertiles for both sPLA2activity and OxPL/apoB had a significantly elevated risk of future CAD,with an OR of 3.46 (2.22-5.42) compared to subjects in the lowesttertiles. A similar but weaker relationship was noted for the highesttertiles of sPLA2 mass and OxPL/apoB (OR 2.39 (1.58-3.61)).

Area under receiver operating curves revealed significantly increasedvalues by adding OxPL/apoB and sPLA2 activity to traditional riskfactors and the FRS (FIG. 4).

Results shown in FIG. 6 are based on 763 cases and 1397 controls whereinall subject characteristics listed in table 1 were available and confirmthat adding OxPL/apoB and sPLA2 activity to traditional risk factors andthe FRS increases the predictive value.

To assess whether oxidative biomarkers provided additional predictivevalue to the FRS, tertiles of OxPL/apoB, sPLA2 mass, sPLA2 activity andLp-PLA2 activity were evaluated within each FRS risk estimate (FIG. 7).In the low FRS categories, the OxPL/apoB measure did not provideadditional predictive value, but sPLA2 mass and activity showed a neartripling of the odds ratio. However, in the high FRS estimates,OxPL/apoB more than doubled the odds ratio for predicting newcardiovascular events, sPLA2 mass and activity was slightly lesspredictive. The combination of OxPL/apoB and sPLA2 activity wasparticularly useful in reflecting risk adjustment among the FRScategories. For example, in the low FRS category, compared to the lowesttertiles, the highest tertiles of OxPL/apoB and sPLA₂ activity wereassociated with an OR (95% CI) of 8.48(2.98-24.16, P<0.001), in themedium FRS category 5.01(2.28-11.41, P<0.001) and in the high FRScategory 14.35(6.2133.17, P<0.001).

This case-control study nested within the prospectively followedEPIC-Norfolk cohort demonstrates that elevated baseline levels ofOxPL/apoB are strongly associated with increased risk of future fataland non-fatal CAD events. Furthermore, increased levels ofphospholipases involved in the metabolism of OxPL, particularly sPLA2activity, potentiated the risk of fatal and non-fatal CAD eventsmediated by either OxPL/apoB.

In the Bruneck study, the hazard ratio of cardiovascular diseasesmediated by OxPL/apoB levels was increased from about 2 to 4 by Lp-PLA2activity (Kiechl et al., Arterioscler Thromb Vasc Biol.2007;27:1788-1795). However, a weak association was noted in thisEPIC-Norfolk study. The explanations are not obvious, but may relate todifferences in methodology (prospective versus case control) or morelikely to the size of the study (82 events in the Bruneck study versus763 events in the EPIC-Norfolk study).

1. A method of identifying a subject having or at risk of having ordeveloping a cardiovascular disease and/or a cardiovascular event,comprising: measuring, in a blood sample obtained from said subject, atleast two cardiovascular risk factors: a) secretory phospholipases A2(sPLA2) activity and b) oxidized phospholipids on apolipoprotein B-100particles (OxPL/apoB), combining said measurements, the combined valueof sPLA2 activity and OxPL/apoB being indicative of having or a risk ofhaving or developing a cardiovascular disease and/or cardiovascularevent.
 2. The method according to claim 1, wherein said combined valueof sPLA2 activity and OxPL/apoB is compared to a reference value.
 3. Themethod according to claim 1, further comprising measuring at least onecardiovascular risk factor selected in the group of Framingham RiskScore (FRS), C-reactive protein (CRP), IgM IC of apoB100 or IgM MDA-LDLand lipoprotein-associated phospholipase A2(Lp-PLA2).
 4. The methodaccording to claim 1, wherein sPLA2 activity, OxPL/apoB and FRS aremeasured.
 5. The method according to claim 1, wherein sPLA2 activity,OxPL/apoB, FRS and CRP are measured.
 6. The method according to claim 1,wherein said cardiovascular disease and/or cardiovascular event isMetabolic Syndrome, Syndrome X, atherosclerosis, atherothrombosis,coronary artery disease, stable and unstable angina pectoris, stroke,diseases of the aorta and its branches (such as aortic thrombosis oraortic aneurysm), peripheral vascular disease, cerebrovascular disease,and any acute ischemic cardiovascular event.
 7. The method according toclaim 1, wherein OxPL/apoB is measured in an immunoassay using anantibody that interacts with OxPL and an antibody that interacts withapoB.
 8. The method according to claim 1, wherein sPLA2 activity ismeasured in a fluorimetric assay using a substrate for sPLA2.
 9. Themethod according to claim 1, for monitoring the efficacy of a treatmentfor a cardiovascular disease.
 10. The method according to claim 2,further comprising measuring at least one cardiovascular risk factorselected from the group consisting of Framingham Risk Score (FRS), CRP,IgM IC of apoB100 or IgM MDA-LDL and LpPLA-2.